AT502722B1 - USE OF INTERFERONE INDUCED GENE 12 (ISG 12, IFI 27) TO MODULATE TRANSCRIPTIONAL ACTIVITIES OF NUCLEAR RECEPTORS - Google Patents

Description

2 AT 502 722 B1

The invention relates to the use of human or mouse ISG12 for the manufacture of medicaments for the treatment of acute or chronic inflammatory diseases, of metabolic diseases or vascular diseases and to a method for diagnosing in vitro the susceptibility of the abovementioned diseases.

The present invention relates to the use of interferon-induced gene 12 (ISG12, IFI27) to regulate transcription activities of nuclear receptors, as well as the use of single nucleotide polymorphisms in the human ISG12 gene as a marker of susceptibility to disease.

Status:

Inflammation is a major factor in vascular occlusive diseases and therefore atherosclerosis can be considered a chronic inflammatory disease. Monocytes, macrophages and THI cells are found in fresh and older lesions responsible for generating specific disease promoting cytokines. In addition to the nuclear factor kappa B {NFkB), the major transcription factor that mediates the inflammatory response, nuclear receptors are also the target for the regulation of a range of cytokines. With respect to nuclear receptors, the inventors and others have been able to demonstrate that NR4A1, as a member of the NR4A family, is regulated by single nuclear receptors in the inflamed vessel, especially endothelial cells, smooth muscle cells and monocytes. In mice that were dominant for negative NR4A1 transgenic mice, a greatly increased degree of restenosis was found in a carotid artery ligation model; also adeno-viral overexpression of wild-type NR4A1 prevented the restenosis process. Both findings indicate a useful role of NR4A1 in vascular disease. Therefore, NR4A1 in conjunction with other nuclear receptors, such as the adapted nuclear receptor PPARa and PPARy, is involved in feedback loops that counteract vascular pathologies. NR4A1 (Nur77) belongs to the NR4A family of nuclear receptors with hitherto unknown ligands for transcriptional regulation. Although prostaglandin A has recently been described as a transactivator of NR4A3, it is believed that the activity of members of the NR4A family is directly dependent on regulatory expression and post-translational modification. Although the activities of co-repressors and co-activators have also been described, none of these regulators was known to be involved in vascular disease. The inventors, therefore, investigated further factors that modulate the activity of these nuclear receptors, which could mitigate their useful effect on vascular disease. US 2005/0009067 A1 discloses the use of the combination of a series of < 3en's, including ISG12 for " gene expression profiling " of pancreatic carcinomas. The "drug screening" described herein refers to the effect of various drugs on the expression profile of the described markers in response to treatment with the appropriate drugs or inhibition of expression of the markers on nRNA or antibody not otherwise defined, and not on the effect of ISG12, which had a hitherto unknown, if any, effect as the target of such drugs, particularly for modifying the activity of transcription factors. The essence of this disclosure relates to a method for characterizing pancreatic tissue, a kit for characterization, and a method for screening pancreatic cells for the effect of test components on changes of 2 or more of the above-identified genes. WO 2004/011618 A1 relates to a method for the identification of genes which are overexpressed in adipose tissue of different mouse strains, as well as the use of the identified genes for the prevention of adipogenesis, for the treatment of diabetes and for the screening of low-molecular substances, adipogenesis modulate or for the treatment of diabetes. One of the genes found is also ISG12, which is not surprising since ISG12 is regulated by interferons, as is known from AT 502 722 B1. Also disclosed herein is a bioassay for identifying substances that can prevent adipose tissue accumulation, exposing cells expressing one of the identified proteins to substances, and then analyzing the cells for changes in their activity.

In document LABRADA, L. et al. Age-dependent resistance to lethal alphavirus encephalitis in mice: analysis of gene expression in the central nervous system and identification of a novel interferon-inducible protective gene, mouse ISG12. Journal of Virology, 2002, Vol. 76, No. 22, pages 11688-11703, describes the differential susceptibility of newborn to four-week-old mice to Sindbis virus infections, and further that ISG 12 is up-regulated in the less sensitive four-week-old mice and that over overexpression of ISG12 in newborn mice is protective against Sindbis virus. JP 2005204549 A describes a method for studying the sensitivity to the progression of an HCV infection. In population studies, SNPs were found in 103 genes that correlate with the progression of HCV infection. Also not surprising is one of the genes ISG12, which is indeed upregulated in viral infections by interferons.

The invention:

The inventors unexpectedly found a new modulator for the activities of some nuclear receptors, namely the interferon-regulated gene 12 (ISG12), which occurs at basic conditions in a low concentration in the nuclear envelope. From the data obtained in vitro, from ISG12 knock-out mice in vivo, and from the frequency of polymorphisms in the ISG12 gene in patients, the inventors concluded that ISG12 is a new gene that is up-regulated in vessels, which in turn increases the nuclear export of useful nuclear receptors and contributes to the downregulation of their transcription activities. ISG12 therefore represents a target for novel therapeutic measures for the treatment of vascular disease. This surprising protective effect of ISG12 has in no way been related in the prior art to the modulation of the activity of transcription factors.

The invention therefore relates, on the one hand, to the use of human or mouse ISG12 for the preparation of medicaments for the treatment of vascular occlusive diseases, atherosclerosis, coronary heart disease, myocardial infarction, angina, stroke, type II diabetes, hypertension and high blood cholesterol.

On the other hand, the invention also encompasses a method for diagnosing in vitro susceptibility for vascular occlusive diseases, atherosclerosis, coronary heart disease, myocardial infarction, angina, stroke, type II diabetes, hypertension and high blood cholesterol, using single nucleotide polymorphisms in the human ISG12 gene is used.

Results:

Finding ISG12:

In the search for NR4A1 interactive proteins, the inventors constructed a probe of a truncated NR4A1 (amino acids 248-557) and used them in a yeast two-hybrid analysis. As one of the positive interaction partners, they found the entire cDNA of the hiterferon-stimulated gene 12 in cytokine-activated, microvascular endothelial cells (ISG12, Eq: 59925613).

The interaction in the yeast was determined by the " Back Transformation " detected (data not shown). The interaction in mammalian cells was demonstrated by co-immunoprecipitating myc-labeled ISG12 with an antibody to NR4A1 that recognizes the endogenous NR4A1 (ATA 1, line 1) and the overexpressed entire NR4A1 (Figure 1 row 2) in 293 cells. Figure 1 shows that NR4A1 reacts with ISG12. Co-immunoprecipitations in 293 cells: NR4A1 was overexpressed in 293 cells in either the absence or presence of ISG12. Lysates from 293 cells were immunoprecipitated for 4 hours with anti-NR4A1 or control IgG; for immunoblotting, a mouse monoclonal anti-myc antibody was used. ISG12 co-precipitated with endogenous and overexpressed NR4A1 (n = 3).

To determine the location in the cell where the interaction takes place, the inventors overexpressed myc-tagged ISG12 and EGFP-tagged NR4A1 in human umbilical vein endothelial cells (HUVECs, Figure 2a). NR4A1 was predominantly localized in the cell nucleus, with ISG12 mainly occurring in the vicinity of the cell nucleus, where it could be observed together with NR4A1 in the convocal laser microscope (yellow ring). These data are consistent with the published localization of ISG12 in the nuclear envelope. By reconstructing the image of the z-stacks in the convocal laser microscope, the common localization of the nuclear envelope could be demonstrated for almost all cell nuclei (data not shown). To demonstrate that co-localization does not occur due to overexpansion of the proteins, the inventors used human umbilical vein smooth muscle (HUASMC) arteries and were able to demonstrate that endogenous NR4A1 is also co-located in the nuclear envelope with over-expressed ISG12 (Figure 2b). , Figure 2.a) showed co-localization of overexpressed NR4A1 with overexpressed ISG12: HUVEC were transfected with EGFP-NR4A1 and myclSG12. After 30 hours, the cells were fixed, immunostained with a monoclonal anti-myc antibody (red fluorescence) and visualized with a convocal laser microscope. In the illustrations shown, the co-located proteins appeared as a yellow ring around the cell nucleus (n = 3). 2b shows the co-localization of endogenous NR4A1 with overexpressed myclSG12 in vascular smooth muscle cells (SMCs): 30 hours after the transfection of SMCs with myclSG12, the cells were fixed, immunostained with anti-NR4A1 (M210, green fluorescence) and anti-NR4A1. myc antibody (red fluorescence) and visualized by the convocal laser microscope. Endogenous NR4A1 is also localized around the cell nucleus together with overexpressed ISG12 (yellow-orange ring, arrow, (n = 2))

Function of ISG12: ISG12 decreases the nuclear localization of NR4A1: In the course of these experiments, the inventors have observed that in the absence of overexpressed ISG12 NR4A1 was found predominantly in the nucleus, while in the presence of overexpressed ISG12 it was also found in the cytoplasm (Fig has been. Figure 3 shows the distribution of overexpressed EGFP-NR4A1 alone in HUVECs and in the presence of overexpressed myclSG12 this was visualized by the convocal laser microscope. NR4A1 (green fluorescence) is located predominantly in the nucleus in the absence of ISG12 (left part), while in the presence of over-expressed ISG12, more NR4A1 was observed in the cytoplasm (right part) (n = 2).

To quantify the effect of ISG12 in the cellular localization of NR4A1, the inventors have analyzed the distribution of NR4A1 between the cytosol and the nucleus by dividing subcellular fractions from 293 cells in which EGFP-labeled NR4A1 in the absence or presence of myc labeled ISG12 were overexpressed. In the presence of ISG12, 20.8 ± 7.4% (mean ± SD of 4 experiments, p <0.05) more NR4A1 was found in the cytosol than in the absence of ISG12. Fig. 4 shows the difference between cytoplasmic and nuclear concentrations of NR4A1, overexpressed alone or together with myclSG12: The relative concentration was determined by Western blots from cell fraction experiments in 293 cells. A representative example will be given. (N = 3)

When the same experiment was performed with an EGFP-labeled NR4A1 construct (AA248-580) that can not be exported from the cell nuclei, no influence of 5 AT 502 722 B1 ISG12 on the subcellular distribution of the construct was observed. (Figure 5) Similarly, the effect of ISG12 on full-length NR4A1 was impaired in the presence of leptomycin B, which prevents nuclear export (data not shown). This indicates that ISG12 is a novel interaction partner of NR4A1 localized in the nuclear envelope and that reduces nuclear localization of the individual nuclear receptors NR4A1 through the mediation of nuclear export. FIG. 5 shows the overexpression of the nuclear export deficient EGFP-dcNR4A1 (248-580) together with myclSG12: The common localization could be observed in HUVECs (yellow ring) around the cell nucleus, but no change in the distribution of the export of missing dnNR4A1 (n = 2). ISG12 reduces the nuclear receptor transcription activities: From this data, the inventors predicted that ISG12 would also affect the transcriptional activities of NR4A1. To test this, the inventors established a luciferase reporter system consisting of fourfold NBRE (NR4A1 monomeric binding site) luciferase construct and analyzed transcriptional activities of NR4A1 in the presence and absence of co-transfected ISG12 (Figure 6, a ). Overexpression of NR4A1 doubled reporter gene activity, while in the presence of overexpressed ISG12 NR4A1-induced regulation of transcriptional activities was conspicuously reduced. This effect was dependent on the amount of ISG12 transferred, as shown by the dose-dependence of the ISG12-mediated inhibition in Figure 6, b. Figure 6 shows that ISG12 downregulated NR4A1 transcription activities: In a series of experiments, the effect of ISG12 overexpression on NR4A1-regulated transcription was evaluated; Luciferase data were normalized to expression of Renilla and are represented as means ± SEM and analyzed by ANOVA. A) Conspicuous (p = 0.000298) downregulation of NR4A1 reporter activity by ISG12 was observed in 293 cells (n = 4). b) Downregulation of NR4A1 reporter activity by ISG12 was dose dependent, as indicated by the effect of different levels of transferred ISG12 in 293 cells; Downregulation of NR4A1 was reduced with decreasing amounts of ISG12 (p = 0.000254, (n = 2)).

To demonstrate that the effect of ISG12 on transcriptional activities of NR4A1 is not restricted to 293 cells and the artificial NBRE construct, the inventors replaced 293 cells with HUVECs and found that enhancement of NR4A1-induced reporter activity was again significant ISG12 was reduced (Figure 7, a); The inventors also performed analogous experiments using HUVECs and a portion of the PAI-1 promoter coupled to luciferase, a construct previously used to demonstrate NR4A1-dependent PAI-1 regulation. As shown in Figure 7, b, PAI-1 reporter activity was upregulated by NR4A1, and more than 8 times in the absence of ISG12, while when ISG12 was co-transfected, again the transcriptional activity of NR4A1 was suppressed. From this data, the inventors concluded that ISG12 not only physically interacts with NR4A1 and alters its predominant nuclear localization, but also conspicuously reduces NR4A1 transcription activities. Fig. 7, a) shows a reporter analysis performed in HUVECs, where it is also shown that the NR4A1 reporter activity of ISG12 was down-regulated (p = 0, 0118, (n = 2)). b) Transcriptional activities of NR4A1 as analyzed using PAI-1 promoter-805- + 20 pUB PAI-1 luciferase construct was also downregulated by ISG12 in HUVECs (n = 3).

In order to hypothesize that ISG12 affects not only the transcriptional activity of NR4A1 but also that of other nuclear receptors, the inventors have again used a luciferase reporter system consisting of triple PPRE (PPAR Respons element) luciferase construct and the transcriptional activities of PPARa and PPARy, in the presence and absence of co-transfected ISG12 (Figure 8). Overexpression of PPARa with RXR increased reporter gene activities more than 50-fold, whereas in the presence of overexpressed ISG12, PPARα-induced upregulation of transcriptional activities was reduced approximately 10-fold significantly (Figure 8, a). A similar effect was observed as PPARa activities in the presence of the specific PPARa 6 AT 502 722 B1

Ligands WY14643 (Figure 8, b) were analyzed. Again, when PPARa was replaced with PPARy, an increase in reporter gene activity in the presence of overexpressed ISG12 was not observed (Figure 8, c), regardless of whether the PPARy binding ROSIGLITAZONE was present (Figure 8, FIG. d) or not. Figure 8 shows that the ISG12 transcriptional activities of PPARa and PPARy transcriptional activities are downregulated, a) The transcriptional activity of PPARα as measured in a reporter analysis in 293 cells transfected with a PPRE reporter construct and RXRa is by Co-transfection with ISG12 conspicuously downregulated (means ± SEM, normalized to Renilla expression, p = 0.000004, (n = 3). B) Experiments as described in a) were performed in the presence of the PPARa ligand WY-14643 (100 μΜ). Also in this case, ISG12 downregulated PPARa-induced reporter activities (p = 0.0000054, (n = 3)). c) Experiments, as in a) using PPARy instead of PPARa, also show that co-transfection with ISG12 significantly (p = 0.0000088) reduces the reporter activities (n = 3), d) the downregulation of PPARy Reporter activities by co-transfected ISG12 were also observed in the presence of the PPARy ligand rosiglitazone (p = 0.0023) in analogous experiments (n = 2). These data indicated that ISG12 not only affects and downregulates the transcriptional activities of NR4A1, but also affects other nuclear receptors by downregulating their transcriptional activities. In fact, a direct interaction of ISG12 with other nuclear receptors could be found during similar experiments using antibodies against RXR or PPAR, with overexpressed myc-tagged ISG12 in 293 cells (Figure 9). Figure 9 shows co-immunoprecipitation of myclSG12 in 293 cells by antibodies to RXRa, NR4A1 and PPARa. Lysates of 293 cells were immunoprecipitated for 4 hours with the respective rabbit antibodies and a preimmune IgG; Western blotting was performed with a mouse monoclonal anti-myc antibody. With all 3 specific antibodies, ISG12 could be precipitated while no specific binding was observed using the pre-immune IgG (n = 2).

Regulated expression of ISG12:

To demonstrate the potential biological role for the interaction of ISG12 with NR4A1 and its inhibitory effect on NR4A1 transcription activities, the inventors have determined the expression of ISG12 in the vasculature. When specimens obtained from human atherosclerotic vascular lesions (defined microscopically) and analyzed by increased expression of II-8 and MCP-1 (Figure 10) for the expression of ISG12 and compared to unaffected contiguous areas, The inventors found in the affected zones an approximately fourfold up-regulation of ISG12. From these data, the inventors concluded that ISG12 is up-regulated in vascular lesions. Fig. 10 shows regulated expression of ISG12 analyzed by QT-PCR; relative expression was normalized to a level of PBGD and the data reported as means ± SEM. ISG12 is up-regulated in human vascular lesions in parallel with the inflammatory marker genes IL-8, MCP-1 (n = 2).

These data indicate that a cytokine or mixture of cytokines is present in the arterial lesions that upregulate ISG12. The inventors have therefore determined the effect of inflammatory cytokine candidates on the regulation of ISG12 in a HUVECs culture (Figure 11). The inventors discovered that of the cytokines analyzed, only interferon-gamma (IFNg) and interferon-alpha (IFNa) ISG12 is up-regulated in endothelial cells, more than 10-fold from low levels at sustained unstimulated conditions. Figure 11 shows that expression of ISG12 in HUVECs is enhanced by stimulation with various inflammatory stimuli (oxPAPC 150 pg / ml, TNFa 100 U / ml, LPS 10 pg / ml, IFNa 1000 U / ml, IFNy 100 ng / ml, TGFβ1 6.6 ng / ml) for 0.5 to 7.5 hours (n = 2).

Generation of mice with missing ISG12: 7 AT 502 722 B1

The ISG12 '' 'mice were generated according to the procedure previously described in the literature and recorded in FIG. The ISG12 + / &quot; mice and cultured male and female ISG ^ 'mice showed no apparent coarse phenotypic pathologies (Figure 12), had normal growth and survival rates and fertility. Routine histological analyzes showed no signs of tissue damage. Fig. 12 shows the method of ISG12 gene deletion. (a) Black boxes in the genomic structure represent exon sequences. Using equivalent recombinations, the new genes replaced a 2.5 kb genomic fragment comprising exons I to IV, resulting in a complete deletion of the ISG12 gene, b) Southern blot analyzes of mouse genomic DNA cleaved with HindIII and analyzed with a 3 external sample, d) ISG12 mice and wild type.

Use of mice with missing ISG12

The inventors used carotid artery ligation to determine the effect of ISG12 gene inactivation on neointimal formations. As shown in Figures 13, a and b, ISG12_ / mice had only insignificant neointimal formations compared to wild-type mice, whereas in non-ligated control carotid arteries no morphological differences were found between wild type and ISG12 '/ _ mice. This suggests that the absence of ISG12 does not affect vascular morphology under baseline conditions where ISG12 is also expressed at low levels in wild type animals. If injury (ligation) induces an upregulation of ISG12, then as in the ISG ^ &quot; Absent animals, the lack of ISG12 is useful for the vascular healing process. Figure 13 shows that no neointimal formations could be observed by the carotid artery ligation model in mice lacking ISG12, a) immunocytochemical image of the carotid wall of wild-type (wt) and ISG ^ mice, ligated and unligated, stained with hematoxylin and eosin b) quantification of morphometer analysis, differences between neointima to media ratio in wt and ISG12 · '' were statistically significant, * p = 0.0038. Another example of the effect of missing ISG12 mice is shown in FIG. Here, the survival of wild type mice due to intraperitoneal injection of endotoxin with ISG 12 + mice is compared. Mice lacking ISG12 show significantly better survival than wild type mice.

Single nucleotide polymorphism in ISG12 is related to vascular disease.

In a human genetics study, the inventors analyzed 3 nucleotide polymorphisms (SNP) in the human ISG12 gene on chromosome 14q (official name: interferon alpha-inducible protein 27 (IFI27), NM_005532). Of these, 2 were polymorphic in our samples (rs2239644 with 15.0% heterosygosity and rs2799 with 4.1% heterozygosity). This polymorphism has been linked to the association with coronary artery disease (recent heart attack or angina), stroke, type II diabetes, arterial hypertension, and hypercholesterolemia in 853 Caucasian individuals performed at the Max Planck Institute of Psychiatry in Munich, Germany, together with a Case study for unipolar recurrent depression analyzed.

The inventors discovered a striking compound of rs2799, which resides in the 3'UTR of the gene, and the presence of hypercholesterolemia (p = 0.006), type 2 diabetes (p = 0.00058), and stroke (p = 0.0008) but not high blood pressure , Heart attacks or angina. All associations remained significant in a logistic analysis when age, gender and depression status were controlled. This suggests that the ISG12 gene may also be important for human vascular disease. Figure 15 shows the distribution of rs2799 genotypes and the presence of vascular risk factors.

Discussion:

The inventors found a new modulator of the transcriptional activities of a number of nuclear receptors, the interferon-regulated gene 12 (ISG12), which interacts with NR4A1. 8 AT 502 722 B1

It is highly regulated by INFa and INFy, and overexpressed in vascular injuries. The inventors further show that here the ISG12, a nuclear envelope protein with hitherto unknown functions, influences the nucleocytoplasmic distribution of NR4A1 and suppresses its transcriptional activity. When mice lacking ISG12 were analyzed for susceptibility to vascular injury, the inventors found that ISG127 mice were resistant to restenosis following carotid artery ligation. When mice with doubled ISG12 and NR4A1 were used, they again showed restenosis on the carotid bowel ligations to a similar extent as wild-type mice. This indicates that in vascular pathologies, the major target for ISG12 is the NR4A1. Further support for a possible importance of ISG12 comes from human genetics studies. The inventors were able to show that there is a strong link between intronic ISG12 single nucleotide polymorphism (SNP), rs2799 and the presence of hypercholesterolemia, type 2 diabetes and stroke. The GG genotype of this polymorphism protects against this disorder. This prompted us to analyze the effect of ISG12 on the assumed nuclear recipes PPARa and PPARy. ISG12 regulates the transcription activities of PPARa and PPARy in a similar way as the transcriptional activity of NR4A1 does. Likewise, ISG12 also interacts physically with these nuclear receptors. These data indicate that ISG12 is a novel gene that is regulated in the vessels in the area of injury and contributes to vascular pathologies by reducing the nuclear export of &quot; useful &quot; Increases nuclear receptors and at the same time reduces the transcription activities. ISG12 will therefore be the target for new therapeutic strategies for the treatment of vascular disease.

methods:

Cell cultures, vectors, transfections and reagents.

Human umbilical vein endothelial cells (HUVECs) and human smooth muscle umbilical artery (HUASMCs) were cultured as previously described and used until the 5th passage. HUVEC was transferred using 1.5 μg of DNA using Lipofectamine Plus® reagents (Invitrogen, Carls-bad, CA) according to the manufacturer's instructions. The cells were treated overnight with M199 (3% serum). Human embryonic kidney 293 cells were cultured as recommended (ATCC, Manassas, VA). 293 cells were transferred for the luciferase reporter assay or for cell fractionation experiments using calcium phosphate and 3 pg DNA.

Human ISG12, full-length wild types and amino acids 1-122, were cloned into pCMV Myc (BD Biosciences-Clontech, Paolo Alto, CA) and NR4A1 into pCMV Myc (total length 1-598) and dominant negative NR4A1 (248-580) was in EGFP-C1 cloned (BD Biosciences-Clontech, Paolo Alto, CA). The luciferase reporter construct NBRE 4x (pGL3 plasmid contains 4 canonical NBRE sides) and pc DNA 3.1 NR4A1 (1-598 amino acids, total length, wt) have already been described. Mammalian expression vectors for human retinoid X receptor alpha (RXRa), mouse PPARy (mPPARy, mouse PPARa (m PPARa), and the luciferase reporter construct PPRE3-TK-LUC32 were provided by L. Nagy, Debrecen, Hungary.

Yeast two-hybrid screening

The yeast 2-hybrid screen was performed using the MATCHMAKER and Screening Kit (BD Biosciences Clontech, Paolo Alto, CA) according to the manufacturer's instructions. Total RNA was isolated from active human uterine microvascular endothelial cells (HUMEC), then stimulated with endotoxin for 4, 9 and 16 hours and tumor necrosis factor alpha (TNFa) for 2, 4, 9 and 16 hours and with trizole (Invitrogen, Carlsbad , CA); mRNA was purified with oligo (dT) -labeled magnetic beads (Dynal, Oslo, Norway). The regulation of II-8 was demonstrated by quantitative rtPCR (Roche Diagnostic, Basel, Switzerland) and confirmed the efficient stimulation after TNF or LPS treatment at the respective time points (data not shown). The mRNAs isolated from the different stimulated cells were pooled 9 AT 502 722 B1 and the first strand synthesis was performed with SMART technology (modified oligo (dT) primers in combination with MMLVRT). The following PCR, cDNAs and linearized pGADT7-Rec vectors containing GAL4 activation domain (AD) were co-transformed to AH109 yeast (MATa, trp1 -901, leu2-3, 112, ura3-52, his3-200, gal4A, gal80A, LYS2: GAL1UAS -GAL 1TATA-HIS3, GAL2UAS-GAL2TATA -ADE2, URA3:: MEL1UAS-MEL1TATA-lacZMELI). For screening, a construct of NR4A2 containing amino acid 248-557 lacking transactivation domain 2 was cloned into pGBKT7 containing the GAL4 binding domain (BD, Paolo ALto, CA)) and the resulting construct was run through Sequencing with ABI Prism Prism® Big Dye ™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) to 310 Genetic Analyzer (Perkin Elmer, Wellesley, MA). Autoactivation was excluded by co-transformation with empty AD. The bait construct was transformed into Y187 yeast chains (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3,112, gal4A, gal80A, met-, URA3 :: GAL1UAS-GAL1TATA-lacZMELI) , "Mating" was performed according to the manufacturer's instructions with an AH109 yeast (microvascular endothelial cell library), (2x106 independent clones [Clontech, Paolo ALto, CA) described by Brondyk.W.H. &Amp; Macara, I.G. Use of two-hybrid system to identify Rab binding proteins. Methods Enzymol. 257, 200-208 (1995)] resulting in approximately 1, € x106 transformants. Positive colony selection was performed on SD medium without leucines, tryptophans, adenines and histidines, and in the presence of 35 mM 3-amino-1,2,4-triazoles (3-AT). Yeast plasmid DNA was prepared by the method of Liang and Richardson [Liang.Q. &Amp; Richardson.T. A simple and rapid method for screening transformant yeast colonies using PCR. Biotechniques 13, 730-2, 735 (1992)], followed by electrotransformations into HB-101 bacteria. The plasmids were isolated from bacteria by Fast-Plasmid ™ mini-kit (Eppendorf, Hamburg, Germany) and by 310 Genetic Analyzer by ABI Prism® Big Dye ™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) sequenced (Perkin Elmer, Wellesley, MA). Positive interactions of "bait and prey" were confirmed by retransformation in yeast.

Immunoprecipitation, Western blotting

The 293 transfected cells were washed with phosphate buffered saline and incubated for 30 min with a cell solution buffer containing 2.7 mM KCl, 1.5 mM KH 2 PO 4, 9.2 mM Na 2 HPO 4-2H 2 O, 150 mM NaCl, 0.7% NP40, 0.3% Triton X-100 and containing complete protease inhibitor cocktail (Roche Diagnostic, Basel, Switzerland). A concentration of NaCl was then set at 350 mM and cell lysates were then incubated for 3 h at + 4 ° with 2 pg of appropriate antibody: NR4A1 M210 (Santa Cruz Biotechnology, Santa Cruz, CA), Anti PPARa H-98 (Santa Cruz Biotechnology , Santa Cruz, CA), antRXRa ΔΝ 197 (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit immunoglobulin fraction (DAKO, Milano, Italy) and protein A-Sepharose beads (Amersham Biosciences, Buckinghamshire, UK). After 5 washes with 1xPBS, then the beads were resuspended in Laemmli buffer.

Immuofluorescence and Confocal Microscopy HUVECs or SMCs were grown to a 50-80% density on coverslips (Nalge Nunc International, Naperville, IL) and with EGFP-NR4A1 (1-598) or EGFP-dominant negative NR4A1 (248-580) and myclSG12 alone or combined transfers. Thirty hours post-transfection cells were fixed in 4% paraformaldehyde, then treated with Triton X-100 and then stained for myclSG12 with anti-myc antibody (Oncogene, San Diego, CA) at a 1: 100 dilution and Alexa Flour 568 conjugated goat anti-mouse second antibody in 1: 200 dilution (Molecular Probes, Eugene, Oregon). The presence of EGFP of the co-transfected vector pEGFP-NR4A1 and pEGFP-dnNR4A1 was determined on a 10 AT 502 722 B1

Olympus AX-70 microscope (HUASMCs) or LSM510 (HUVECs) microscope (Zeiss, Germany) at 488 nm excitation and 512 nm emission wavelength and distinguished from the Alexa Flour 568 label (excitation wavelength 543 nm, emission wavelength 603 nm).

cell fractions

Human embryonic kidney cells (293) were grown in a 6-well subconfluency and then transfected with EGFP-NR4A1 and myclSG12 together or separately. The cell fractions were treated 30 hours after transfection using an MS buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl, pH 7.5 and 1 mM EDTA) containing 1% protease inhibitor cocktail. After 10 times homogenizing the cells with a 27 'needle, the samples were centrifuged at 1300xg for 10 min at 4 ° C to pellet the cell nuclei. The supernatant, containing cytoplasmic and heavy membrane fractions, was decanted and centrifuged again at 16,000xg for 30 min to separate the cytoplasm; then the membranes were pelleted. Core pellets were washed again with ice-cold PBS and resuspended in MS buffer. The nucleus was purified by a second centrifugation at 1300xg and then lysed from the nuclei by a buffer containing: 2.7mM KCl, 1.5mM KH 2 PO 4, 9.2mM Na 2 H PO 4-2H 2 O, 150mM NaCl, 0.7% NP40, 0.3% Triton XM. 100 and complete protease inhibitor cocktail (Roche Diagnostic, Basel, Switzerland). Separated nuclear and cytoplasmic fractions were dissolved in Laemmli buffer and blotted after separation on a 9% SDS PAGE Western using an anti-EGFP antibody (Abcam, Cambridge, UK).

Luciferase analysis:

Human embryonic kidney cells (293) or HUVECs were transferred as mentioned above with Renilla vector as an internal control. In the case of experiments in which PPARs were stimulated by their binding, the medium was changed 9 hours after transfection and stimulation was performed with WY-14 643 (Biomol, Hamburg, Germany) or Rosigli-tazone Maleate (Alexis Biochemicals, Lausen, Switzerland ) Performed 21 hours before the luciferase or Renilla analyzes. Luciferase and Renilla analyzes were performed 30 hours after transfection of the cells in cell lysates with a dual luciferase assay kit (Promega, Madison, Wisconsin) according to the manufacturer's description. The luciferase activity was normalized to the respective renilla values. All experiments were performed in triplicate or at least in duplicate. RNA isolation and relative, quantitative reverse transcriptase polymerase chain reaction (Q-PCR) RNA from stimulated HUVECs and human and mouse tissue samples were obtained using Trizol (Invitrogen, Carlsbad, CA) reagents. Total RNA (900 ng) was reverse transcribed with MuLV reverse transcriptase using the Gene Amp-RNA PCR kit (Applid Biosystems, Foster City, CA) and oligo dT16 primer. Isolated mouse arterial tissue was immersed in ice-cold RNAIater (Ambion, Austin TX) and stored in buffers at -70 ° until isolation. RNA isolation of pooled arterial tissue (n = 3) was performed as described by Furnkranz.A. et al. Oxidized phospholipids. trigger atherogenic inflammation in murine arteries. Arterioscler. Thromb. Vase. Biol. 25, 633-638 (2005), and 350 ng RNA were reverse transcribed with the same kit as before.

The mRNA sequences for gene analysis were obtained from GenBank. The primers (mouse and human) were constructed using PRIMER3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). The following forward (F) and reverse primers (R) were used for human ISG12: F, 5'-TGTGATTGGAGGAGTTGTGG -3 '; R, 5'-GAACTTGGTCAATCCGGAGA -3 '; Mouse ISG125'-CTG CCA TAG GAG GAG CTC TG-3 'and 5'-ATG GCA TTT GTT GAT GTG GA-31; human MCP-1; Mouse MCP-1; human IL-8 F, 1 1 AT 502 722 B1 5'-CTC TTG GCA GCC TTC CTG ATT-3 '; R, 5-TAT GCA CTG ACA TCT AAG TTC TTT AGC A-3 '. Q-PCR was performed by LightCycler technology using Fast Start SYBR Green I kit for amplification and detection (Roche Diagnostics, Basel, Switzerland). In all analyzes cDNA was reduplicated by a standard program (10 'denaturation steps and 55 cycles of 5' at 95 ° C; 15 'at 65 ° C and 15' at 72 ° C; melting point analyzes in 0.1 ° C increments; cooling step). Each Light Cycler capillary was loaded with 1.5 μL MgC12 (25 mM); 10.1 pLH20; and 0.4 pL of each primer (10 pM). The final amount of cDNA per reaction refers to 2.5 ng of total RNA used for the reverse transcription. The relative quantification of target gene expression was performed using Pfaffl mathematical models. Expression of the target molecule was standardized to expression of PBGD with primers for human PBGD F, 5'-TCG AGT TCA GTG CCA TCA TC-3 'and PBGD R, 5-CAG GTA CAG TTG CCC ATC CT-3' or mouse PBGD ,

Generation of ISG 12 ^ and mice with ISG12 "/ _ NR4A1" / 'double deficiency

The ISG mice were cultured according to the method described by Uhrin, P. et al. Disruption of the protein C inhibitor results in impaired spermatogenesis and male infertility. J. Clin. Invest 106, 1531-1539 (2000). described procedure generated. The murine gene consists of 4 exons separated by 3 relatively small introns (introns I to III). The cDNA sequence of mlSG12 was used to construct primers specifically for the different regions of mlSG12 genes. Using 129S / v genomic DNA as template, the PCR reactions were optimized and used for screening (HindIII from a 129S / v mouse genomic DNA) and cloned into a RPCI.22 BAC vector (Invitrogen, Carlsbad , CA). The genomic clones thus obtained were used to prepare the target vectors that inactivate the ISG12 gene in embryonic stem cells (ES). A total of 8.5 kb homologous sequences from the ISG12 gene were introduced into a parental pPNT vector containing neomycin phospho-transferase (neo) cassettes and a herpes simplex virus pPNT.mlSG12. contains. In the first step, a 4.7 kb Xhol fragment (containing 5'UTR of mlSG12 gene) was joined to an XhoI site of a pPNT-generated plasmid pPNT1.mlSG12. Next, a 3.8 kb EcoRI fragment (comprising 3'UTR of a mlSG12 gene) was cloned to the EcoRI site of pBluescript® II KS (+/-) vector (Stratagene, La Jolla, Ca). Subsequently, a 3.85 kb Sall-Smal fragment from this vector was Klenow-filled and joined to Asp718 site / Klenow-filled pPNT1.mlSG12, in which the vector pPNT.MISG12 was taken. Transfection of target vectors resulted in 4 out of 200 G418 / ganciclovir double-resistant clones undergoing the desired homologous recombination as determined by extensive Southern blotting of the isolated genomic DNA derived from R1 embryonic stem cells (ES) , confirmed by the 129S7v mouse chain (obtained from A. Nagy, Samuel Lunenfeld Institute, Toronto, Canada).

Chimeric mice (F0) obtained from 8 cell stage embryo aggregates of ES cell clones were test grown for germline transmission with Swiss mice. They transferred the destroyed ISG12 alleles to their origin (50% 129S / v: 50% Swiss genetic background) by obtaining ISG12 + / 'mice. Intercrossings of these mice resulted in ISG ^ 'mice as identified by Southern blot analyzes of tail-tip DNA using the 3' external probe. The correct inactivation of the ISG12 genes was further confirmed by additional reviews using 5 'external, 5' internal, neo-specific and 3 'flanked internal samples (not shown) and by RT-PCR. Wild type and heterozygous control mice used in the experiment were taken from a litter of the corresponding knockouts. All experiments were carried out in accordance with the institutional instructions and reviewed by the respective university committee (Austrian approval for animal experiments No 169/1999).

In order to generate ISG ^ 'double-deficient mice and the corresponding control mice with single 1 2 AT 502 722 B1 ISG ^' or NR4AT / 'deficiencies, ISG ^' mice were crossed with NR4A1 '/' mice (in B6 background, supra) Provided by J. Milbrandt of the Department of Pathology, Washington University School of Medicine, St. Louis, USA). The resulting double-heterozygous mice were then cultured to obtain ISG12 + / + NR4A1 + / +, ISG12 + / + NR4A1 '/', ISG12_ / 'NR4A1 + / + and ISG12_ /' NR4A1 '/' mice. These mice were used for analysis. Genotyping the mice for the NR4A1 alleles was performed by Southern blotting using an exon-2 specific probe and BamHI cleaved genomic DNA (a 4.9 kb and a 6.6 kb fragment for the wild types and recombinant DNA, respectively). ,

Animal experiments: For the restenosis mouse model, 8-week-old mice were used for a carotid artery ligation [published method of Kumar.A. &Amp; Lindner.V. Remodeling with neointimal formation in the mouse carotid artery after cessation of blood flow. Arterioscler. Thromb. Vase. Biol. 17, 2238-2244 (1997)]. The left common caretid artery of ketamine / xylazine anesthetized mice was ligated near the distal bifurcation (n = 4 to 7). 2.5 or 4 weeks after ligation, the mice were anesthetized and subsequently the heart pumped with PBS and the carotid arteries removed. For the atherosclerotic lesion model in mice, apo E + and wt mice were anesthetized with ketamine / xylazine at 6 months of age and then pumped through the heart with PBS and the aortic arch and cardiac membrane removed.

morphometry

The artery was cut from the ligature to the aortic arch, fixed with 4% paraformaldehyde and then stained with hematoxylin and eosin or with smooth muscle actin (clones 1A4-FITC, Sigma) and counterstained with DAPI (4,6 diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA). A standardized reference point was set in which the vascular structure was not deformed by the ligature and the lamina elastica were intact. Sections 0.7 mm, 1.7 mm and 2.7 mm from the reference point were morphometrically analyzed using the AnalySiS® Software-Pakt (Soft Imaging System; Münster, Germany) for digital images of the vessels (green channel: smooth muscle actin, blue channel : DAPI and Red Channel: Autofluorescence of the Lamina Elastica), obtained with an F-View camera on an Olympus AX-70 microscope. The circumference of the lumen, the internal elastic lamina and the external elastic lamina were measured and the media area, neointimal area and neointima / media ratio were calculated.

Patient recruitment 853 individuals were recruited at the Max Planck Institute of Psychiatry in Munich (Germany) in the context of a case study of unipolar recurrent depression, 88% of whom were German and the remainder of Caucasian descent. In these patients, the psychiatric diagnosis was determined by WHO mandate according to DSM-IV using the Clinical Evaluations in Neuropsychiatry (SCAN) plan. Appropriate controls were selected by a Munich-based collective and screened for the presence of anxiety and conditioned depression using the Composite International Diagnostic Screener. In addition, 35 different types of medical, neurological and psychiatric depression were evaluated in all depressed and healthy subjects and their first-degree relatives using a self-description. For this study, diseases such as myocardial infarction or angina, stroke, high blood pressure, high blood cholesterol and type 2 diabetes were documented in the patients and the respective controls and used for statistical analysis. Of the 853 people, 366 had unipolar depression, 35 had coronary artery disease or angina, 161 had high cholesterol, 1 had hypertension, 37 had diarrhea and 11 had strokes. 67.3% were female and the mean age was 50.8 years (SD = 13.8).

genotyping

At the beginning of this study, 40 ml of EDTA blood was taken from each patient and examined. The DNA was extracted from fresh blood using Puregene ™ whole blood DNA extraction kit (Gentra Systems Inc, MN). 3 SNPs (rs223944, rs223945 and rs2799) were selected from human chromosomes of ISG12, IFI27 (NM_005532) located on chromosome 14q32 (using dbSNP) (http://www.ncbi.nlm.nih.gov: 80 /). The SNP search tool at http://ihg.gsf.de/ihg/snps.html was used to download SNP sequences from public records. Hary Weinberg Equilibrium (HWE) was tested in the psychiatric control group. Both SNPs were found in Hardy Weinberg Equilibrium in this group of individuals (p = 0.20 for rs2799 and p = 0.44 for rs223944).

Genotyping was performed on a MALDI-TOF mass spectrometer (MassArray® system) using the Spectrodesigner software (Sequenom ™ CA) for primer selection and multiplexing, and the homogenetic mass extension (hMe) was processed for the production of primer extension products. Genotyping was performed at Geneticx Research Center GmbH, Munich, Germany. All primer sequences are available on request.

Statistical analyzes

Experimental values are given as an average ± SEM. The statistical significance of differences in the results were analyzed by Student's unpaired two-tailed t-test.

The significance of differences in morphometric analysis was determined using Mann-Whitney 2-tailed non-parametric U-test and expressed as a probability score. IFI27 SNPs have been tested for association with myocardial infarction or angina, stroke, high blood pressure, high blood cholesterol and type 2 diabetes. Analyzes for these "case-control" relationships were performed using the Armitage's trend test as a statistical method at (http://ihg.gsf.de/cgi-bin/hw/hwa2.pl). The statistical tests were submitted for input to the Internet by Sasieni [Sasieni, P.D. From genotypes to genes: doubling the sample size. Biometrics 53, 1253-1261 (1997)]. In addition, link analyzes were performed by calculating logistic regression using SPSS for Windows (Releases 11, SPSS Inc., Chicago, DL) genotype-level test effects with age, gender, and depression status as value pairs.

GenBank accession numbers:

Protein Homo sapiens ISG12, Eq: 55925614; Mus musculus ISG12 EI: 44771124; Homo sapiens Nur77 GL21361342, mRNA Homo sapiens ISG12 GL55925613; Mus musculus ISG12 EI: 44771123; Homo sapiens Nur77 GL21361342 mRNA Homo sapiens Hydroxymethylbilane synthase HMBS (PBGD) GI: 66933007mRNA Homo sapiens Interleukin 8 (IL8) Eq: 28610153 Target for new therapeutic and diagnostic strategies in inflammation, metabolic and vascular diseases.

Claims (2)

Claims 1. Use of human or mouse ISG12 for the preparation of medicaments for the treatment of vascular occlusive diseases, atherosclerosis, coronary heart disease, myocardial infarction, angina, stroke, type II diabetes, hypertension and high blood cholesterol. 2. A method for diagnosing in vitro the Suszeptibilät for vascular occlusive diseases, atherosclerosis, coronary heart disease, myocardial infarction, angina, stroke, diabetes type II, hypertension and high blood cholesterol, characterized in that single nucleotide polymorphisms in the human ISG12 gene is used. Including 15 sheets of drawings